The present invention relates generally to an apparatus and method for growing and applying specific aerobic and facultatively anaerobic cultures of bacteria such as Pseudomonas Fluorescens, Bacillus Subtilis, Bacillus Licheniformis, Bacillus Thuringiensis, Starkeya Novella and sulfur metabolizing bacteria to soil bioremediation, biological pest management and wastewater treatment processes including carbonaceous BOD removal, grease digestion, denitrification, odor control, methane gas production, pond clarification and the restoration of the percolation rates in media such the soil that has clogged biomat. A principal application for this device is the biological reduction of nitrate contamination. The device has broad application where bacteria are used in industrial, agricultural, forestry and bioremediation processes.
There are many applications where bacteria may be used beneficially in residential, commercial, and industrial applications. Non-Pathogenic bacteria enhance either directly or indirectly through enzyme production many activities in all of society. In the area of wastewater treatment the benefits of creating an environment conducive to the growth and reproduction of beneficial bacteria has long been known. In other areas, such as the prevention of freezing and frost, it is being learned that bacteria can perform tasks heretofore expected of chemical agents. In nearly all cases the method has been to produce bacteria in an industrial setting and apply those bacteria on a continual basis or to adjust the environmental conditions and depend upon natural selection to have the correct species of bacteria grow on site.
A. Wastewater:
Wastewater contains pathogenic bacteria, carbonaceous compounds, nitrogenous compounds, odorous sulfur compounds, and grease. All of these pollutants have been traditionally stabilized with biological treatment processes. Many species of native bacteria accomplish the stabilization process in conventional secondary wastewater treatment. Biological nitrogen removal is an important aspect of wastewater treatment. Findings confirm the existence of a diverse community of heterotrophic bacteria in addition to nitrosomonas, nitrobacter, and nitrosococcus, which are involved in nitrogen removal during wastewater treatment. Of these heterotrophic bacteria, Pseudomonas spp. are known to be dominant denitrifiers and show significant involvement in both nitrification and denitrification processes. Pseudomonas bacteria have demonstrated direct oxidation of ammonium to nitrates with little or no nitrite accumulation. Both Pseudomonas and Bacillus spp. show significant involvement in nitrification. Soil bacteria such as Pseudomonas Fluorescens, Bacillus Subtilis and Bacillus Licheniformis to name three, are superior degraders of both carbon and nitrogen compounds. These soil bacteria are present in wastewater treatment processes in diminutive numbers along with many other species. Many of the other species originate from the human gut and are not aggressive degraders of carbon. These include coliform group bacteria. The soil bacteria that are cultured by the method and device described, do not naturally predominate in wastewater treatment processes as they do in soil. The device and methods are used to establish these bacteria as dominant cultures in such wastewater treatment processes.
In aerobic conditions the heterotrophic nitrifying bacteria pseudomonas Fluorescens can use an enzyme called ammonia monooxygenase (AmoA) to oxidize ammonia to hydroxylamine, nitrite, and nitrate with a small but significant release of nitrite and nitrate. The ammonia monooxygenase found in pseudomonas is thus similar to the amoA gene found in autotrophic ammonia oxidizers such as nitrosomonas europaea. The nitrate is reduced to nitrite and further reduced to nitric oxide (NO) under anaerobic conditions. Ref. (Daum M, Zimmer W, Papen H, Kloos K, Nawrath K, Bothe H. “Physiological and molecular biological characterization of ammonia oxidation of the heterotrophic nitrifier Pseudomonas putida.” Curr Microbiol 1998 October; 37(4):281-8. Medline) Protein and other nitrogenous compounds are likewise broken down by respective enzymes. The stepwise pathway involved is as follows. In aerobic conditions Pseudomonas converts ammonia into nitrite and nitrate which is then reduced as the effluent flows into the anaerobic zone, and begins to act as an electron acceptor and oxidize carbon while being reduced by the denitrification pathway from Nitrate>Nitrite>Nitric oxide>Nitrous oxide>Nitrogen gas with the consumption of carbon into carbon dioxide or cell matter. In this process some of the carbon for denitrification is obtained through metabolic consumption of what is called the zoogleal biomat, an anaerobic assemblage of bacteria and other organisms that form a humic filter.
Approximately 25% of the US population relies upon decentralized wastewater treatment. This number is growing. Many systems are old and were not constructed in a way that offers the maximum protection of ground water. In these systems it is common for nitrogen to escape the treatment zone and enter the ground water.
The design of standard septic treatment and disposal systems relies upon anaerobic conditions in the septic tank and throughout the leach trench up to and inclusive of the biomat. Anaerobic conditions exist until the effluent passes through the biomat and hopefully enters into an aerobic treatment zone. In a standard anaerobic septic treatment system ammonia and reduced forms of nitrogen pass completely through the system almost unaltered. They are converted to oxides of nitrogen like nitrite, and nitrate after escaping the biomat and leaving the treatment zone. Under this form of treatment nitrate may enter the ground water and contaminate drinking water supplies. Nitrate in drinking water poses a health threat.
A further concern in onsite waste treatment is the fact that biomat growth tends to clog soil and creeping failure occurs as the soil percolation rate diminishes and effluent rises in the trench. This causes one common type of leach field failure, the surfacing of effluent. Unable to percolate and in excess of transpiration demand, the effluent will pool on the surface, following the occluded side walls of the trench. Such pooling brings humans and animals into contact with pathogens. This anaerobic biomat is sensitive to air and to the attack by aerobic bacteria and invertebrates such as nematodes. Aerobic bacteria are normally not present or long lived in the biomat. They are excluded except at the outside interface where predation by worms, nematodes, bacteria, and fungi occurs. Pseudomonas bacteria stimulate the predation of biomat by these invertebrate organisms and participate in the competitive inhibition on several levels. They encourage the invertebrate grazers and produce antibiotic compounds that competitively inhibit certain other bacteria such as the slime producing coliform.
The wastewater treatment described in U.S. Pat. No. 4,279,753 entitled “Wastewater Treatment System Including Multiple Stages of Alternate Aerobic-Anaerobic Bioreactors in Series” to Nielson et al., is confined to improving and enhancing a natural biological process that removes suspended, dissolved organic matter and nitrogen from the wastewater. This is accomplished by a series of alternating aerobic-anaerobic bioreactors with the effluent stream contacting microorganism located within the bioreactors.
The process described in U.S. Pat. No. 4,042,458 entitled “Process For The Production Of Micro-organisms” to Harrison is specifically designed to improve the anaerobic digestion process by increasing the number of Methylococcus bacteria. This process involves placing pure strains of Pseudomonas into the process and does not involve growing the bacteria at the site of use.
The process described in U.S. Pat. No. 4,999,111 entitled “Process For Treating Water” to Williamson details a modification to the activated sludge process of treating wastewater. This modification is specifically designed to improve the removal of Phosphorus from the wastewater through the adjustment of environmental factors.
The method described in U.S. Pat. No. 6,383,390 entitled “Method Of Treating Ammonia-comprising Wastewater” to VanLoosdrecht, et al. reveals a two stage process for treating ammonia in a waste stream. The environmental conditions of the stages are adjusted and no microorganisms are added to either stage.
The process described in U.S. Pat. No. 6,447,681 entitled “Aquaculture Wastewater Treatment System And Method Of Making Same” to Carlberg, et al. is a three phase system specifically for treating waste from fish culture. The system utilizes macroorganisms, a traditional ammonia removal system, and constructed wetlands for treatment before dispersal. The system does not introduce any outside microorganisms.
The system described in U.S. Pat. No. 6,497,819 entitled “Method and Apparatus For Treating Wastewater” to Baba, et al. discloses a device to be put into direct contact with the wastestream for the treatment of that wastestream. It utilizes a macromolecular substance to house the microorganisms which provide treatment. The system does not utilize any selection or inoculation.
B. Petroleum Hydrocarbon Contamination of Soil and Water:
Petroleum hydrocarbon originating from refineries, crude oil drilling operations, pipeline breaks, leaking underground storage tanks, and spills on land and sea, are a major source of pollution. The bacterial formulation described in U.S. Pat. No. 5,531,898 entitled “Sewage And Contamination Remediation And Materials For Effecting Same” to Wickham discloses use of bacteria including: Pseudomonas Fluorescens, Bacillus subtilis, and Bacillus licheniformis. These are, in the case of pseudomonas, aerobic and, in the case of bacillus, facultative anaerobic heterotrophic bacteria. These bacteria have been shown to digest crude oil, diesel, BTEX, and most forms of TPH. They have applications in the bioremediation of soil and aquatic petroleum contamination as a biodegrader, a source of enzymes for cleaving hydrocarbons. Rapid or accelerated bioremediation of major petroleum contamination sites, requires enormous numbers of these bacteria. A method for such quantities was not readily available, and this resulted in increased cost to end users. In order to produce large numbers of these bacteria rapidly it was necessary to develop an aerobic bacteria generator such as the device and method described in this patent.
The microorganism and method described in U.S. Pat. No. 6,521,444 entitled “Microorganism And Method For Environmental Purification Using The Same” to Numata, et al. is a novel microorganism which has been altered to allow it to be efficient at decomposing trichlorethlyne. It is a very specific microorganism which serves a very specific purpose.
The process described in U.S. Pat. No. 6,569,333 entitled “Restoring Soil And Preventing Contamination Of Groundwater” to Takagi, et al. describes a method to selectively grow bacteria on agar. The bacteria are then mixed with a porous media which is then mixed with the soil to be treated. The porous material traps contaminants and water flow until treatment is completed.
The method described in U.S. Pat. No. 6,368,019 entitled “Method for soil remediation” to Sugawa, et al. reveals a process to inject liquid containing a specific group of microorganisms into the earth near a site contaminated with hydrocarbons. The injection of the liquid forces volatile components out of the soil pores and they are captured at the surface. The microorganisms effect treatment of the small amount of remaining contaminant. The microorganisms are not grown at the site, but manufactured in an industrial setting.
C. Phytophora, Pythium Damping Off, Bacterial, Viral, and Fungal Plant Diseases
Phytophora infestans was the cause of late blight of potatoes and was responsible for the Irish potato famine of 1845. The organism grows on leaves. The disease, which can destroy a field crop within days, causes mottled, dark lesions on leaves and stems from which develop a white, velvety growth that kills the plant. Blighted potatoes develop a dark, corky rot and appear dehydrated. It is a virulent and contagious disease. Hyphae grow between the cells thrusting haustoria into neighboring cells and also grow through stomates of leaves which then develop into branched sporangiophores. Raindrops help spread sporangia to other plants. One variety is now responsible for a recent outbreak of sudden oak death in the United States caused by Phytophthora ramorum. This disease attacks oak, bay, Douglas fir, redwood, rhododendron, madrone, grape, and many other valuable fruit, timber and ornamental species.
Pseudomonas fluorescens and Pseudomonas corrugata have been tested as biocontrol agents against Pythium damping off of sugar beet. Incorporation into the seed coat offers a practical way of applying the biocontrol agents. For optimization of biocontrol, the determination of the minimum initial dose necessary for successful biocontrol is crucial. (Schmidt, C. S.; Agostini, F.; Mullins, C. m.; Leifert, C., Influence of Initial Antagonist Dose on Sugarbeet Root Colonization and Biocontrol of Pythium Damping Off, University of Aberdeen, Department of Plant and Soil Science, and Aberdeen University Centre for Organic Agriculture (AUCOA), Aberdeen UK)
Antagonistic performance of Pseudomonas fluorescens increases with dosage. Doses larger than 107 CFU/seed pellet are necessary to inhibit Pythium damping off disease. Conversely, antagonistic performance of Pseudomonas corrugata follows an optimum curve. Numbers of healthy plants as well as plant fresh and dry weight reach highest levels when 104-106 CFU/seed pellet are applied and these indicators decrease at higher doses. The ratio between applied bacteria (CFU/seed pellet) and recovered bacteria per plant clearly shows that the applied Pseudomonas strains not only persist but also propagate on the seedling surfaces. Both Pseudomonas strains are able to build up large populations (1-3×105) on sugarbeet seedlings even when low initial doses (103 CFU/seed pellet) are applied. At low doses, up to 80-300 fold more cells than initially applied can be recovered. At doses exceeding 105-106 CFU/seed, however, the total population per seedling of the Pythium damping off antagonist does not increase, and, in fact, the number of cells recovered decreases compared to initial dose. Thus, a saturation point appears to be reached with 105-106 CFU/plant. Population sizes of both antagonists reach maximum levels (>104 CFU/cm) at the hypocotyl and the upper parts of the root (0-2 cm below seed level) already when the lowest dose is applied (103 CFU/seed pellet). Measurable bioluminescence indicates high metabolic activity of the strains in the hypocotyl and the upper parts of the root at all applied doses. In Pseudomonas corrugata, population size does not increase with dose at all whereas in Pseudomonas fluorescens a slight, but significant increase of the total population per plant with dose is observed, due to an increased colonization of the lower root parts (exceeding 4 cm root depth). Not only the population size but also differences in the velocity of the population build up and in antibiotic production at different initial doses may account for the observed significant effects of dose on biocontrol efficacy. Downward colonization of sugarbeet roots by Pseudomonas fluorescens is significantly increased in five different soils by combining it with Bacillus subtilis in a mixed inoculum. (Schmidt, C. S.; Agostini, F.; Whyte, J.; Simon, A. M.; Mullins, C. M.; Leifert. C., Influence of Soil pH. Soil Temperature and Soil Type on Biocontrol of Pythium Damping Off Disease by Antagonistic Bacteria, University of Aberdeen, Department of Plant and Soil Science, and Aberdeen University Centre for Organic Agriculture, Aberdeen UK).
The material and method revealed in U.S. Pat. No. 4,952,229 entitled “Plant Supplement And Method For Increasing Plant Productivity And Quality” to Muir details a soil supplement to be manufactured. The supplement consists of a specific mix of microorganisms which are designed to improve plant growth. The supplement is applied in the solid form.
The process described in U.S. Pat. No. 5,507,133 entitled “Inoculant Method And Apparatus” to Singleton, et al. is designed for the specific purpose of growing rhizobia to inoculate legumes. The system utilizes prepackaged units with two compartments: one containing peat moss as a substrate and the other containing pure cultures of rhizobia. The units are shipped to the site of inoculation, mixed, and inoculation takes place after a growth period.
The device described in U.S. Pat. No. 6,432,698 entitled “Disposable Bioreactor For Cultivating Microorganism And Cells” to Gaugler, et al. describes a system for the production of a specific nematode organism to be used as a biological pesticide. The system is designed to be shipped to a site and the product utilized after an incubation period. The system utilizes no aqueous phase.
D. Hog, Dairy and Aquaculture.
Bacillus subtilis is used in aquaculture for applications such as the larval rearing of the white shrimp Penaeus schmitti. In this application there are two benefits. Bacillus subtilis is useful in controlling the gut epithelium scaling syndrome of Penaeus schmitti, known as “Bolitas.” This has beneficial effects upon survival, metamorphosis rate, larval quality and size of the postlarval shrimp. The antibiotic activity permits a reduction in the daily water exchange rate from 100% to 30% in the larval rearing process.
The process described in U.S. Pat. No. 4,927,751 entitled “Process For Obtaining Exoenzymes By Culture” to Memmer, et al. reveals a two step process which uses a highly complex fermentation unit. This process is limited to the production of enzymes only.
The process described in U.S. Pat. No. 5,283,059 entitled “Process For The Producing A Stabilized Spore Forming Viable Microorganism Preparation Containing Bacillus Cereus” to Suzuki, et al. provides for the growth of specialized bacteria in a starch solution. This solid mixture is then pelletized and used as animal feed.
The method described in U.S. Pat. No. 5,967,087 entitled “Method Of Increasing Seafood Production In The Barren Ocean” to Markels, Jr. relates to the addition of fertilizer to ocean areas. This addition of fertilizers and iron chelates increases seafood production by stimulating aquatic plant growth. No microorganisms are grown utilizing this system.
The process described in U.S. Pat. No. 6,183,739 entitled “Phospholipase In Animal Feed” to Baudeker, et al. involves a method to increase feed utilization in animals by adding phospholipase to the animal feed.
In each of the cases cited above the system described either modifies the environment, provides an additive, or adds microorganisms to a process. In no case is there a system which provides for the continual growth of specific microorganisms at a site where they are needed. The systems depend upon a laboratory to provide large quantities of microorganisms which are at a much higher purity than needed for many uses.
There remains a need for a simple device that can be inoculated with bacteria and will grow bacteria for inoculation into an in situ process or treatment. Further, the need exists for a method to provide these bacteria in quantity over long periods of time. There is a further need for microorganisms to be produced in an aqueous solution to allow for ease of handling.